Immunoblotting

What is the purpose of Electrophoretic transfer in Immunoblotting?

Allow protein movement

What is the purpose of adding milk protein or BSA to the incubation buffer in immunoblotting?

Blocking agent to prevent antibodies (proteins themselves) to stick to free membrane non-specifically. -immobilizes ...

Why are secondary antibodies useful?

Economically efficient (only need a limited amount of the flurochore antibody (specific) then can buy a cheaper batch of the non specific antibodies (have to be a different species). Amplification of signal

What is the colorimetric method?

Immunofluorescence->important enzyme is present on second antibody but substrate is everywhere on blot, so the reaction from substrate to product (color) from enzyme is on the POI (antibody)

What is chemiluminescence?

Immunofluoresence- > substrate produces a product and during this reaction, light is emitted. (recorded by a movie)

What is the equation of Kd?

Kd = [P] [L] / [PL] (PL = protein-ligand complex)

Which term do we use to describe the affinity of a binding pair?

Km

What is the definition of Kd?

The concentration of ligand at which half of the binding sites are saturated. The lower the Kd the less ligand needed to saturate half of the binding sites the tighter the binding

Why would the antibodies stick to the free membrane?

The free membrane, or nitrocellulose membrane, is a very nonspecific membrane that causes proteins (such as antibodies) to stick to it.

How does free energy of the protein changes due to affinitiies?

The larger the free energy change when two molecules bind, the stronger the interaction.

Why must the secondary antibody be of a different species than the primary antibody?

an anti-mouse injected in goat will elicit a response since sees protein as a foreign object, so goat produces antibodies that bind to the anti-mouse.

What is the reaction catalyzed by alkaline phosphataste (colorimetric assays)

when phsohphate is on the 5-bromo... molecule, it cannot react with Nitroblue. With alkaline phosphatase (AP), phosphate is removed then the two molecules can interact and indigo color is shown.

How can we detect where our antigen-antibody-antibody sandwich is located?

1) Colorimetric 2) Chemiluminescence 3) Fluorescence

What is the structure of an antibody (Immunoglobulin G, IgG)?

2 heavy chains 2 light chains Connected by disulfied bonds 2 antigen binding sites ~150,000 Da (g/mol)

What is the purpose of ethanol in Immunoblotting?

Addition of ethanol helps to strip SDS from the proteins after removing them from the gel and enhances protein binding to the membrane.

After staining the separated proteins of SDS PAGE with Coomassie blue, what can be seen?

All proteins will be stained blue Intensities of bands can be compared between lanes Protein standard ladder will be used to deduce protein sizes

What are two ways to visualize proteins after SDS PAGE?

Coomassie staining and immunoblotting

What is the difference in use between coomassie staining and immunblotting?

Coomassie staining of all proteins and immunoblotting to visualize only the protein of interest

What is Kd?

Dissociation constant-gives us a measure of binding strength

How can we see a particular protein on a gel?

Immunoblotting AKA Western Blot

What is fluorescence?

Immunofluorescence--> dye molecules are attached to 2nd antibody. Use wavelength that excites the dye molecule and it will emit a different wavelength to be seen.

Are proteins equally accessible to antibodies when inside the plyacrylamide gel?

No- pore size, large proteins trouble moving, (ask teacher)

Proteins won't initially be visible on the gel of SDS PAGE. What can you do to visualize these separated proteins?

Stain ALL proteins on the gel, add Coommassie blue dye (blue form only)

What does affinity of a non-covalent interaction between two binding partners mean?

The strength of the binding

What does TTBS stand for?

Tween 20 TRIS (buffer region 7=9 pH) Buffered Saline (Na+Cl-)

How does Western Blotting, or immunoblotting work?

We use a specific antibody that only recognizes the protein of interest to bind to it and visualize this binding.

If we didn't use blocking agents (casein or BSA), what would happen to our immunoblot results?

We would have a lot of background and we would not be able to detect specific targeting of our POI.

What is Electrophoretic transfer?

horizontal electric field applied between SDS PAGE and porous membrane.

What is the set up of electrophoretic transfer?

negative ---> SDSPAGE ---> Porous Membrane ---> positive